ASK1 and p38 MAP kinase regulate proteolytic processing of ENaC

Abstract

These Ussing chamber studies were undertaken to investigate the mechanism by which hydrogen peroxide (0.5 mM) inhibits mouse epithelial Na+ channels (ENaC) transfected into FRT cell monolayers.We found the inhibition was blocked by expression of a dominant negative construct of the kinase ASK1, and was replicated by expression of a constitutively active mutant of ASK1. The effects of both hydrogen peroxide and of the constitutively active mutant of ASK1 were blocked by the p38 MAP kinase inhibitor, SB203580, and by expression of a dominant negative mutant of p38 MAP kinase.Western blotting with an antibody for phosphorylated p38 MAP kinase confirmed that ASK1 modulates the amount of phosphorylated p38 MAP kinase. Epithelial Na+ channels are regulated by the Nedd4–2 system that controls their surface expression. We found, using surface biotinylation methods, however, that neither ASK1 nor p38 MAP kinase affected surface expression of the channels.Epithelial Na+ channels are normally activated by proteolysis of the α‐ and γ‐subunits during their intracellular processing. When we used ENaC subunit constructs in which the N‐ and C‐terminals are labelled with V5 or FLAG tags to examine the effect of the kinases on proteolytic processing, we found that they inhibit proteolytic cleavage of both α‐ and γ‐ENaC.In summary, we have found that hydrogen peroxide inhibits epithelial Na+ channels expressed in FRT cell monolayers via the kinases ASK1 and p38 MAP kinase, which reduce the activity of the channels by inhibiting the normal activation of the channels by proteolysis.